Generating tailor-made enzymes to study biological processes and to catalyze useful new reactions remains one of the most exciting prospects of chemical biology. The rational design of enzymes with novel activities has generally proven to be difficult, Hedstrom, et al, Science 1992, 255, 1249-53, however, because our understanding of the interactions that govern protein function is not yet sufficiently sophisticated to predict reliably the effects of perturbing a protein's primary structure. Mimicking methods used by Nature to produce proteins with biologically essential activities, molecular evolution provides an alternate approach to generating enzymes with new functions. This approach involves iteratively (i) diversifying a protein of interest into a large library of mutant proteins, typically using whole genome mutagenesis, Schimenti, et al., Genome Res. 1998, 8, 698-710; Cupples, et al., Proc. Natl. Acad. Sci. USA 1989, 86, 5345-9; Hart, et al., J. Am. Chem. Soc. 1999, 121, 9887-9888, random cassette mutagenesis, Reidhaar-Olson, et al., Methods Enzymol. 1991, 208, 564-86; Reidhaar-Olson, et al., Science 1988, 241, 53-7, error-prone PCR, et al., PCR Methods Applic. 1992, 2, 28-33, or DNA shuffling, Stemmer, Nature 1994, 370, 389-91; Minshull, et al., Curr. Opin. Chem. Biol 1999, 3, 284-90; Harayama, Trends Biotechnol. 1998, 16, 76-82; Giver, et al., Curr. Opin. Chem. Biol. 1998, 2, 335-8; Patten, et al., Curr. Opin. Biotechnol. 1997, 8, 724-33, (ii) screening or selecting these variants for proteins with desired activities, and (iii) amplifying the genetic material (usually DNA) encoding the evolved proteins.
While a number of proteins have been evolved successfully using this strategy, the scope of protein molecular evolution is currently limited by the small number of methods to screen or select for proteins with desired properties. Among these methods, in vivo selections, in which cells expressing proteins with desired new functions propagate exponentially while cells expressing undesired library members fail to grow, offer several important advantages over in vitro selections and over both in vitro and in vivo screens. Because each molecule in an in vivo selection does not need to be individually separated and assayed, as is the case in screens, the potential diversity of proteins explored by in vivo selections is limited only by the transformation efficiency of E. coli. In vivo selections can therefore process protein libraries that are approximately 10, Hoseki, et al., J. BioChem. (Tokyo) 1999, 126, 951-6, members and thus 1,000- to 1,000,000-fold larger than protein libraries that are screened. Unlike selections performed in vitro, which typically select for binding or for a single bond-forming or bond-breaking event, Jäschke, et al., Curr. Opin. Chem. Biol. 2000, 4, 257-62; Famulok, et al., Curr. Opin. Chem. Biol. 1998, 2, 320-7; Pedersen, et al., Proc. Natl. Acad. Sci. USA 1998, 95, 10523-8, in vivo selections can choose proteins based on their ability to catalyze multiple-turnover reactions in the more relevant context of a living cell. Despite these considerable advantages, very few in vivo selections for proteins with desired properties exist. The vast majority of the in vivo selections described to date fall into one of two categories. Most link cell survival to a protein's function through complementation of an essential biosynthetic enzyme. Yano, et al., Proc. Natl. Acad. Sci. USA 1998, 95, 5511-5; Altamirano, et al., Nature 2000, 403, 617-22. The major limitation of this approach, however, is that proteins of interest can only be evolved to catalyze naturally occurring and metabolically critical reactions. The second major type of in vivo selection used for protein evolution selects for proteins that can transform substrates into the sole carbon source available to the cell. Membrillo-Hernandez, et al., J. Biol. Chem. 2000, 275, 33869-75; Bornscheuer, et al., Bioorg Med. Chem. 1999, 7, 2169-73. This selection is limited, however, to those enzymes that process cell permeable substrates into forms of carbon that can be processed by the cell.
In addition to suffering from a lack of more general in vivo selections prior strategies for the molecular evolution of proteins have been also limited by a lack of methods to select against undesired specificities or activities. As a result, evolved enzymes typically exhibit broadened, rather than truly altered, specificities or activities, Fong, et al., Chem. Biol. 2000, 7, 873-83; Iffland, et al., Biochemistry 2000, 39, 10790-8; Jurgens, et al., Proc. Natl. Acad. Sci. USA 2000, 97, 9925-30; Lanio, et al., J. Mol. Biol. 1998, 283, 59-69; Kumamaru, et al., Nat. Biotechnol. 1998, 16, 663-6; Zhang, et al., Proc. Natl. Acad. Sci. USA 1997, 94, 4504-9; Liu, et al., Proc. Natl. Acad. Sci. USA 1997, 94, 10092-10097; Yano, et al., Proc. Natl. Acad. Sci. USA 1998, 95, 5511-5, in contrast to the exquisite substrate specificities and precise activities that are characteristic of natural enzymes. Broadened specificities can emerge because the determinants allowing acceptance of a new substrate are often not mutually exclusive with those that allow acceptance of the wild-type substrate.
The lack of methods to select against undesired activities also prevents the evolution of a second important feature of many natural enzymes, the ability to be active under one set of conditions but inactive under slightly different conditions. Developing methods for the evolution of conditionally active proteins would enable researchers to address fundamental questions in protein function. For example, evolving a protein that is active in the presence of an exogenously added cell-permeable small molecule but inactive in the absence of this small molecule would allow for the first time the study of how an allosteric binding site can evolve in a library of closely related enzymes. The evolution of allostery would also reveal how frequently small molecule binding sites emerge during protein diversification.
Enzymes that manipulate the covalent structure of proteins and nucleic acids are of particular interest to chemists and biologists. These enzymes play important roles in biological processes ranging from the insertion of viral DNA into a host's genome to post-translational processing of essential enzymes. In addition, many of these enzymes catalyze intrinsically interesting and powerful chemical processes such as amide bond rearrangement or the cleavage and ligation of DNA with single-site per genome specificity. Finally, many enzymes that manipulate the structures of proteins and nucleic acids have proven to be extremely useful in a wide range of research applications including protein chemical synthesis, Chong, et al., Gene 1997, 192, 271-81; Blaschke, et al., Methods Enzymol. 2000, 328, 478-96; Evans, et al., Biopolymers 1999, 51, 333-42; Severinov, et al., J. Biol. Chem. 1998, 273, 16205-9; Muir, et al., Proc. Natl. Acad. Sci. USA 1998, 95, 6705-10, protein purification, Chong, et al., Gene 1997, 192, 271-81; Evans, et al., J. Biol. Chem. 1999, 274, 18359-63; Mathys, et al., Gene 1999, 231, 1-13, protein engineering, Ayers, et al., Biopolymers 1999, 51, 343-54; Holford, et al., Structure 1998, 6, 951-6, genome mapping, Thierry, et al., Nucleic Acids Res. 1992, 20, 5625-31; Belfort, et al., Nucleic Acids Res. 1997, 25, 3379-88; Copenhaver, et al., Plant J. 1996, 9, 259-72; Liu, et al., Proc. Natl. Acad. Sci. USA 1996, 93, 10303-8; Mahillon, et al., Gene 1997, 187, 273-9; Mahillon, et al., Gene 1998, 223, 47-54, screening protein libraries, Daugelat, et al., Protein Sci. 1999, 8, 644-53, and the creation of conditional genomic knock outs. Le, et al., Methods Mol. Biol. 2000, 136, 477-85; Rajewsky, et al., J. Clin Invest 1996, 98, 600-3; Yoon, et al., Gene 1998, 223, 67-76; Yoon, et al., Genet. Anal 1998, 14, 89-95. For these reasons, recombinases, homing endonucleases, and inteins have been the focus of intense research efforts over the past several years. Effective systems for generating and characterizing altered versions of these enzymes, however, have not been developed.
There remains a need for the development of improved systems for characterizing protein variants. There is a particular need for the development of systems that allow in vivo selection of protein activity. There is also a need for the development of systems that allow the characterization of altered biological cleavage proteins, and particular for the identification of cleavage enzymes with altered specificity.